Treatment of autoimmune disease

ABSTRACT

The present invention features a novel combination therapy useful in the treatment of autoimmune disease that increases or maintains the number of functional cells of a predetermined type in a mammal by killing or inactivating autoimmune cells and re-educating the host immune system.

CROSS REFERENCE TO RELATED APPLICATIONS

More than one reissue application has been filed for the reissue of U.S.Pat. No. 6,660,487. The reissue applications are application Ser. Nos.12/911,587 (the present application) and 10/851,983, which is nowReissue U.S. Pat. No. Re. 41,887. This application is a continuationreissue application of reissue application Ser. No. 10/851,983, filedMay 21, 2004, now U.S. Pat. No. Re. 41,887, which is a reissue of U.S.Pat. No. 6,660,487, which is a continuation-in-part of Ser. No.09/521,064, filed on Mar. 8, 2000, now U.S. Pat. No. 6,599,710, which,in turn, claims benefit of U.S. provisional application Serial No.60/123,738, filed on Mar. 10, 1999, botheach of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Early onset diabetes mellitus, or Type I diabetes, is a severe,childhood, autoimmune disease, characterized by insulin deficiency thatprevents normal regulation of blood glucose levels. Insulin is a peptidehormone produced by the β cells within the islets of Langerhans of thepancreas. Insulin promotes glucose utilization, which is important forprotein synthesis as well as for the formation and storage of neutrallipids. Glucose is also the primary source of energy for brain andmuscle tissue. Type I diabetes is caused by an autoimmune reaction thatresults in complete destruction of the β cells of the pancreas, whicheliminates insulin production and eventually results in hyperglycemiaand ketoacidosis.

Insulin injection therapy has been useful in preventing severehyperglycemia and ketoacidosis, but fails to completely normalize bloodglucose levels. Although insulin injection therapy has been quitesuccessful, it does not prevent the premature vascular deteriorationthat is the leading cause of morbidity among diabetics today.Diabetes-related vascular deterioration, which includes bothmicrovascular deterioration and acceleration of atherosclerosis, caneventually cause renal failure, retinal deterioration, angina pectoris,myocardial infarction, peripheral neuropathy, and atherosclerosis.

A promising treatment for diabetes, islet transplantation, has been inhuman clinical trials for over ten years. Unfortunately, the resultswhere Type I diabetes is the underlying etiology are poor. There havebeen many successes with islet transplantation in animals, but onlywhere the animals are diabetic due to chemical treatment, rather thannatural disease. The only substantiated peer reviewed studies usingnon-barrier and non-toxic methods and showing success with islettransplants in naturally diabetic mice use isogeneic (self) islets. Theisogenic islets were transplanted into already diabetic NOD micepre-treated with TNF-alpha (tumor necrosis factor-alpha); BCG (bacillusCalmette-Guerin, an attenuated strain of mycobacterium bovis); and CFA(complete Freund's adjuvant), which is an inducer of TNF-alpha(Rabinovitch et al., J. Immunol. (1997)159(12):6298-303). This approachis not clinically applicable primarily because syngeneic islets are notavailable. In the allograft setting of islet transplantation, the graftsare rejected presumably due to autoimmunity. Furthermore, diabetic hosttreatments such as body irradiation and bone marrow transplantation aretoo toxic in Type I diabetes patients, rendering the short-termalternative of insulin therapy more attractive.

I previously developed a transplant method to introduce allogeneic andxenogeneic tissues into non-immunosuppressed hosts, in which the cellsare modified such that the donor antigens are disguised from the host'simmune system (Faustman U.S. Pat. No. 5,283,058, hereby incorporated byreference). Generally, masked islets or transgenic islets with ablatedclass I are only partially protected from recurrent autoimmunity inspontaneous non-obese diabetic (NOD) mice (Markmann et al.,Transplantation (1992) 54(6):1085-9). There exists the need for atreatment for diabetes and other autoimmune diseases that halts theautoimmune process.

SUMMARY OF THE INVENTION

The present invention provides a novel method for reversing existingautoimmunity.

Accordingly, the invention provides a method for increasing ormaintaining the number of functional cells of a predetermined type(e.g., islet cells) in a mammal, involving the steps of: (a) providing asample of cells of the predetermined type, (b) treating the cells tomodify the presentation of an antigen of the cells that is capable ofcausing an in vivo autoimmune cell-mediated rejection response, (c)introducing the treated cells into the mammal, and (d) prior to, after,or concurrently with step (c) treating the mammal to kill or inactivateautoimmune cells of the mammal.

In preferred embodiments, step (b) involves eliminating, reducing, ormasking the antigen, which is preferably is MHC class I. Such methodsare known, and are described, e.g., in Faustman, U.S. Pat. No.5,283,058.

Preferably, step (d) involves administering to the mammal tumor necrosisfactor-alpha (“TNF-alpha”), or a TNF-alpha inducing substance, (i.e., anagonist). As will be explained in more detail below, the TNF-alphasignaling pathway is an inflammatory pathway that effectively bringsabout killing of the autoimmune cells that attack the desired cells.There are many methods for stimulating TNF-alpha production, includingthe following: vaccination with killed bacteria or toxoids, e.g., BCG,cholera toxoid, or diphtheria toxoid; induction of limited viralinfections; administration of LPS, interleukin-1, or UV light;activation of TNF-alpha producing cells such as macrophages,B-lymphocytes and some subsets of T-lymphocytes; or administration ofthe chemotatic peptide fMET-Leu-Phe; CFA-pacellus toxoid, Mycobateriumbovis bacillus, TACE (a metalloproteinase that mediates cellularTNF-alpha release), hydrozamates, p38 mitogen activated protein (“MAP”)kinase, and viral antigens that activate NF_(κ)B transcription factorsthat normally protect the cells from apoptosis (i.e., cell death).

Killing of undesired autoimmune cells can also be accomplished byadministering agents that act as agonists for the enzyme, TNF-alphaconverting enzyme, that cleaves the TNF-alpha precursor to producebiologically active TNF-alpha.

Autoimmune cells can also be killed by administering agents that disruptthe pathways that normally protect autoimmune cells from cell death,including soluble forms of antigen receptors such as CD28 onautoreactive T cells, CD40 on B cells that are involved in protection ofautoimmune cells, and CD95 (i.e., Fas) on T-lymphocytes. Other suchagents include p75NTF p75TNF and lymphotoxin Beta receptor (LtbetaR).

The methods of the invention in some respects run counter to currenttreatment regimens for autoimmune diseases. Many of the major approvedtherapies for such diseases involve the administration ofanti-inflammatory drugs that inhibit the production of TNF-alpha,including COX-2 inhibitors, and TNF antagonists. My studies indicatethat these conventional therapies are actually deleterious, in that theybring about expansion of the population of harmful autoimmune cells inthe patient, increasing the number and severity of lesions andautoreactive infiltrates. In addition, many of these anti-autoimmuneinflammatory drug therapies cause severe re-bound disease afterdiscontinuation. For example, treatment with anti-inflammatory agentsactually increases the number of lymphocyte infiltrates in the pancreasof a diabetic. Once treatment is discontinued, these lymphocytes regaintheir normal function, resulting in a heightened autoimmune response.

The methods of the invention can be used to treat any of the major HLAclass II-linked autoimmune diseases characterized by disruption in MHCclass I peptide presentation and TNF-alpha sensitivity. These diseasesinclude, for example, type I diabetes, rheumatoid arthritis, SLE, andmultiple scelorosis. The method can be used in any mammal, e.g., humanpatients, who have early pre-symptomatic signs of disease, or who haveestablished autoimmunity.

The invention also provides a method for increasing or maintaining thenumber of a predetermined type e.g., islet cells, in a mammal by thesteps of (a) treating the mammal with an agent that kills or inactivatesautoimmune cells of the mammal; (b) periodically monitoring the celldeath rate of the autoimmune cells; and (c) periodically adjusting thedosage of the agent based on the information obtained in monitoring step(b).

In any of the methods of the invention in which TNF-alpha isadministered or stimulated, two agents can be used together for thatpurpose, e.g., TNF-alpha and IL-1 can be used in combination therapy, ascan any other combinations of agents.

In addition, the invention provides a method for diagnosing anautoimmune disease or predisposition to such a disease in a mammal(e.g., a human patient). The method includes the steps of (a) providingperipheral cells from a mammal; (b) treating these cells with aTNF-alpha treatment regimen and; (c) detecting cell death in theperipheral cells, where an increase in cell death, when compared withcontrol cells, is taken as an indication that the mammal has anautoimmune disease or predisposition to such disease. This diagnosticmethod can be used for any mammal, however, the preferred mammal is ahuman patient. Peripheral cells that can be used in this method are, forexample, splenocytes, T lymphocytes, B lymphocytes, or cells of bonemarrow origin and in step (b) of the method, these cells are preferablytreated with TNF-alpha.

By “functional cell,” is meant cells that carry out their normal in vivoactivity. In certain preferred embodiments of the invention, it ispreferred that the cells are capable of expressing endogenous selfpeptide in the context of MHC class I.

By “predetermined type,” when used in reference to functional cells, ismeant that one may select a specific cell type. For example, one skilledin the art may decide to carry out the method of the present inventionin order to increase or maintain the number of functional islet cells inthe pancreas. In this example, the predetermined cell type is isletcells.

By “class I and peptide” is meant MHC class I presentation of peptide(i.e., self peptide) on the cell surface. Cytoplasmic antigens arebelieved to be processed into peptides by cytoplasmic proteases and atleast in part by the proteasome, a multicatalytic proteinase complex ofwhich the Lmp2 protein, discussed herein, is associated. The process ofMHC class I presentation is thought to include formation of a complexbetween the newly synthesized MHC class I molecule, including aglycosylated heavy chain non-covalently associated withβ2-microglobulin, and peptide within the rough endoplasmic reticulum ofthe cell. Thus, “class I and peptide” refers to the MHC class I/peptidecomplex as it is presented on the cell surface for education of theimmune system.

By “killing” or “kills” is meant to cause cell death by apoptosis.Apoptosis can be mediated by any cell death pathway. According to thepresent invention, cells that are susceptible to killing are defectivein protection from apoptosis due to a defect in a cell death pathway.

“Autoimmune cells,” as used herein, includes cells that are defective inprotection from apoptosis. This defect in protection from apoptosis canbe in the pathway linked to TNF-induced apoptosis, or an apoptoticpathway unrelated to TNF. Autoimmune cells of the present inventioninclude, for example, adult splenocytes, T lymphocytes, B lymphocytes,and cells of bone marrow origin, such as defective antigen presentingcells of a mammal.

By “defective” or “defect” is meant a defect in protection fromapoptosis.

By “exposure” is meant exposure of a mammal to MHC class I and peptide(i.e., self peptide or endogenous peptide) by any means known in theart. In one preferred embodiment, exposure to MHC class I and peptide iscarried out by administering to the mammal an MHC class I/peptidecomplex. In other preferred embodiments, exposure to MHC class I andpeptide occurs by exposing the mammal to cells that express MHC class Iand peptide.

By “cells capable of expressing MHC class I and peptide” is meant, forexample, cells that are class I⁺ or cells that are class I^(−/−) (e.g.,cells having a mutation in the β₂M gene) but that are reconstituted invivo by a compensatory component (e.g., serum β₂M).

By “maintenance of normal blood glucose levels” is meant that a mammalis treated, for example, by insulin injection or by implantation of aeuglycemic clamp in vivo, depending on the host being treated.

By “lmp2 gene or an equivalent thereof,” is meant a cell that has adefect in prevention of apoptotic cell death, for example, a cell thathas an ablation at a critical point in an apoptotic cell death pathway.In another aspect, “lmp2 gene or an equivalent thereof” means that acell has a mutation in the lmp2 gene or a gene that carries out afunction the same as or similar to the lmp2 gene (i.e., a gene encodinga proteasome subunit). Alternatively, the phrase “lmp2 gene or anequivalent thereof” can be used to refer to a cell that has a mutationin a gene that encodes a regulator of the lmp2 gene or another componentof the proteasome complex. For example, a human homolog of the murinelmp2 gene is an equivalent of the lmp2 gene according to the presentinvention. As but another example, a gene that carries out the same orsimilar function as the lmp2 gene, but that has a low amino acidsequence similarity, would also be considered as an equivalent of thelmp2 gene according to the present invention.

“Combination therapy,” or “combined therapy,” as used herein, refers tothe two-part treatment for increasing the number of functional cells ofa predetermined type that includes both (1) ablation of autoimmunecells, and (2) re-education of the host immune system.

By “TNF-alpha induction,” “TNF-alpha treatment regimen,” or “TNF-alpha”includes the administration of TNF-alpha, agents that induce TNF-alphaexpression or activity, TNF-alpha agonists, agents that stimulateTNF-alpha signaling, or agents that act on pathways that causeaccelerated cell death of autoimmune cells, according to the invention.Stimulation of TNF-alpha induction (e.g., by administration of CFA) ispreferably carried out prior to, after, or during administration (viaimplantation or injection) of cells in vivo.

By “effective,” is meant that the dose of TNF-alpha, or TNF-alphainducing agent, administered, increases or maintains the number offunctional cells of a predetermined type in an autoimmune individual,while minimizing the toxic effects of TNF-alpha administration.Typically, an effective dose is a reduced dose, compared to dosespreviously shown to be ineffective at treating autoimmune disease,particularly established autoimmune disease.

The methods of the invention provide, for the first time, effectivereversal of naturally-occurring (as opposed to chemically induced)mediated diseases such as type I diabetes.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiment thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows three graphs that depict blood glucose concentration atindicated times after transplantation (left panels) and six photographsshowing the histology of the pancreas (middle panels) and graft siteunder the kidney capsule (right panels) of diabetic NOD female micesubjected to transplantation with islets from various donor types and asingle injection of CFA. Islet grafts were derived from young NOD mice(panel A), C57 mice (panel B), or β₂M^(−/−) C57 mice (panel C).

FIG. 2 is a graph depicting the histological characteristics of thegraft site and pancreas of individual NOD hosts subjected totransplantation of islets from various types of donors in the absence orpresence of TNF-alpha induction. Open squares indicate lack of visibleislet structures and of visible lymphocytic accumulation; open squareswith dots indicate massive lymphocytic accumulation obscuring isletremnants; shaded squares indicate viable islets without lymphocytes;shaded squares with dots indicate viable islet structures with onlycircumferential lymphocytic accumulation; panc indicates pancreas.

FIG. 3 shows five graphs depicting blood glucose levels (left panels)and five photographs showing the histology of the pancreas (rightpanels) of diabetic NOD female mice subjected to transplantation withislets from β₂M^(−/−) C57 mice and a single injection of CFA. Arrowsindicate the time of removal of the kidney containing the islet graft bynephrectomy.

FIG. 4 shows two graphs (panels A and B) and three photographs (panelsC, D, and E) that demonstrate the effect of TNF-alpha induction andrepeated exposure to C57 splenocytes on islet regeneration andrestoration of normoglycemia in diabetic NOD hosts. Panel A representsNOD females treated with daily injections of insulin alone (controls,n=5). Panel B represents NOD females treated with insulin (untilnormoglycemia was restored) plus a single injection of CFA and biweeklyinjections of 9×10⁶ C57 splenocytes (n=9). Arrows represent time ofdeath. Pancreatic histology of a control animal (panel C); an animalthat remained hyperglycemic (panel D); and an animal in whichnormoglycemia was restored (panel E).

FIG. 5 shows four graphs (left panel) that depict the effect ofmaintenance of normoglycemia during TNF-alpha induction and splenocytetreatment on islet regeneration in diabetic NOD mice. The graphs areaccompanied by eight photographs that show the histology of thepancreas, specifically islets and associated lymphocytic infiltrates(middle panels) and islet insulin content (right panels). Arrowsrepresent time of removal of euglycemic clamp. Mice received a singleinjection of CFA only (panel A), CFA plus biweekly injections ofsplenocytes (9×10⁶) from normal C57 mice (panel B), β₂M^(−/−),TAP1^(−/−) C57 mice (panel C), or MHC class II^(−/−) C57 mice (panel D).

FIG. 6 shows six graphs depicting flow cytometric analysis of the effectof islet regeneration on the percentage of CD3⁺ T cells amongsplenocytes of NOD mice. Percentage CD3⁺ cells is shown in the upperright corner of each graph. Panel A represents a 6- to 7-month-oldfemale C57 mouse; panel B represents a diabetic NOD female treated withinsulin alone for 12 days; panels C through F represent diabetic NODfemales implanted with a euglycemic clamp for ˜40 days and treated witha single injection of CFA either alone (panel D) or together withbiweekly injections of normal C57 splenocytes (panel C), MHC classII^(−/−) C57 splenocytes (panel E), or β₂M^(−/−) , TAP1^(−/−) C57splenocytes (panel F).

FIG. 7 shows the effects of TNF-alpha on the survival of spleen cellsderived from untreated C57 (C57BL/6) and untreated or treated NOD mice.Spleen cells were incubated without or with TNF-alpha (20 ng/ml) for 24hours after which apoptotic cells were detected by flow cytometry withfluorescein isothiocyanate-conjugated annexin V. The percentage ofapoptotic cells is given in the top right-hand corner of each panel.

FIG. 8 shows the blood sugar levels in adoptive transfer recipients offresh untreated spleen cells (top) or in vitro TNF-alpha treated cells(bottom) from diabetic NOD mice transferred to young irradiated male NODmice. In the top panel, mice were injected with 2×10⁷ spleen cellspooled from three spontaneously diabetic NOD mice. In the bottom panel,recipient mice were also injected with spleen cells pooled fromthree,spontaneously diabetic NOD mice. Prior to injection, thesesplenocytes were treated with 10 ng/ml TNF-alpha in vitro and assayedfor cell viability. 2×10⁷ viable spleen cells were then injected intorecipient mice.

DETAILED DESCRIPTION

The present invention provides a method of increasing or maintaining thenumber of functional cells of a predetermined type in a mammal bypreventing cell death. In preferred embodiments, this method is used totreat an autoimmune disease where endogenous cell and/or tissueregeneration is desired. Such autoimmune diseases include, withoutlimitation, diabetes melitus mellitus, multiple sclerosis, prematureovarian failure, scleroderm scleroderma, Sjogren's disease, lupus,vilelego, alopecia (baldness), polyglandular failure, Grave's disease,hypothyroidism, polymyosititis, pempligus pemphigus, Chron's Crohn'sdisease, colititis, autoimmune hepatitis, hypopituitarism,myocardititis, Addison's disease, autoimmune skin diseases, uveititis,pernicious anemia, hypoparathyroidism, and rheumatoid arthritis. Oneaspect of the invention provides a novel two-part therapeutic approachto ablate existing autoimmunity while re-educating the immune system viaMHC class I and peptide. A key feature of the invention is the discoverythat reexpression of endogenous antigens in the context of class I MHCis essential to terminate an ongoing autoimmune response.

As mentioned above, Type I diabetes results from destruction of thecells of the Islet of Langerhans of the pancreas via a severe autoimmuneprocess. The goal for treatment of Type I diabetic patients is topermanently halt the gaff autoimmune process so that pancreatic isletsare preserved. Alternatively, in cases where islet destruction fromautoimmunity is complete, the goal is to provide a method of replacingislet cells, or allowing them to regenerate. Thus, the inventionprovides a novel method for increasing or maintaining the number offunctional cells of a predetermined type for treatment of establishedcases of diabetes melitus mellitus, where existing autoimmunity isreversed.

In adult onset diabetes, or Type II diabetes, the β islet cells of thepancreas are often defective in secretion of insulin. However, recentstudies indicate that, in some patients, autoimmune destruction of βislet cells does play an important role in disease progression (Williset al., Diabetes Res. Clin. Pract. (1998) 42(1):49-53). Thus, thepresent invention may also be used to treat Type II diabetes where anautoimmune component is present.

Relating the Present Invention to Known Genetic and FunctionalInformation

Genetic and functional studies have identified mutations in the lmp2gene in NOD diabetic mice, a murine model for human type I diabetes (Liet al., Proc. Natl. Acad. Sci., USA (1994) 91:11128-32; Yan et al., J.Immunol. (1997) 159:3068-80; Fu et al., Annals of the New York Academyof sciences (1998) 842:138-55; Hayashi et al., Molec. Cell. Biol. (1999)19:8646-59). Lmp 2 is an essential subunit of the proteasome, amulti-subunit particle responsible for processing a large number ofintracellular proteins. The pronounced proteasome defect in Lmp2 resultsin defective production and activation of the transcription factorNF_(κ)B through impaired proteolytic processing of NF_(κ)B to generateNF_(κ)B subunits p50 and p52 and impaired degradation of the NF_(κ)Binhibitory protein, I_(κ)B. NF_(κ)B plays an important role in immuneand inflammatory responses as well as in preventing apoptosis induced bytumor necrosis factor alpha (TNF-alpha). Autoreactive lymphoid cellsexpressing the lmp2 defect are selectively eliminated by treatment withTNF-alpha, or any TNF-alpha inducing agent, such as complete Freund'sadjuvant (CFA), or an agent that acts on a pathway required for celldeath protection, for example, any pathway converging on the defectiveapoptotic activation mechanism. This is well illustrated by faultyapoptosis protection in the NOD mouse which lacks formation ofprotective NF_(κ)B complexes.

The lmp2 gene is genetically linked to the MHC locus (Hayashi et al.,supra). Antigen presenting cells of NOD mice cease production of LMP2protein at approximately 5-6 weeks, a process that terminates the properprocessing of endogenous peptides for display in the context of MHCclass I on the cell surface. Surface display of endogenous peptide inthe context of MHC class I molecules is essential for the selectiveelimination of T cells reactive to self antigens (Faustman et al.,Science (1991) 254:1756-61; Ashton-Rickardt et al., Cell (1993)73:1041-9; Aldrich et al., Proc. Natl. Acad. Sci. USA (1994)91(14):6525-8; Glas et al., J. Exp. Med. (1994) 179:661-72). Currenttheory suggests that interruption of endogenous peptide presentation viaMHC class I prevents proper T cell education and is responsible for adiverse array of autoimmune diseases (Faustman et al., supra; Fu et al.,J. Clin. Invest. (1993) 91:2301-7). These data are also consistent withthe clear sex-, tissue-, and age-specific differences in the expressionof this error which parallel the initiation and disease course ofinsulin-dependent (type I) diabetes. It is hypothesized that the triggerfor the initiation of autotimmunity is the tissue- anddevelopmental-specific dysregulation of the proteasome (or MHC class I)in islet cells, as opposed to lymphocytes. As mentioned above, it ispossible that this defect triggers a pathologic T cell response to isletcells via interruption of proper T cell education (Hayashi et al.,supra).

In a normal, non-diabetic, human or animal, peripheral tissues,including islets, consistently express endogenous antigens in thecontext of MHC class I (Hayashi et al., supra). Constitutivetissue-specific display of self peptide via MHC class I could maintainperipheral tolerance in the context of properly selected lymphocytes(Vidal-Puig et al., Transplant (1994) 26:3314-6; Markiewicz et al.Proceedings of the National Academy of Sciences of the United States ofAmerica (1998) 95(6):3065-70). In the absence of such tissue-specificdisplay, poor negative selection of T-lymphocytes could lead tooverexpansion of self-reactive lymphocytes, a prominent feature in humanand murine disease models.

As mentioned above, autoreactive lymphoid cells expressing the lmp2defect are selectively eliminated, for example, by treatment withTNF-alpha, or any TNF-alpha inducing agent, such as complete Freund'sadjuvant (CFA). Although the specific gene defect has not beenidentified in human autoimmune patients, it is known that humansplenocytes in the human diabetic patient, like murine splenocytes inthe NOD mouse, have defects in resistance to TNF-alpha induced apoptosis(Hayashi et al., supra). Specific cells in human autoimmune patientsmight express a genetic defect, similar to the proteasome defect inmice, that increases susceptibility to TNF-alpha induced apoptosis or ananalogous apoptotic cell death pathway. Therefore, in patientsexpressing the genetic defect, only the autoimmune cells are killed.Permanently eliminating the autoreactive cells is a key feature of aneffective treatment for an autoimmune disease.

According to the present non-limiting theory, of the invention, multiplecell death pathways exist in a cell and any one or more of these celldeath-related pathways may be defective, accentuating the sensitivity ofthese cells to cell death. For example, susceptibility to TNF-alphainduced apoptosis could occur via a failed cell death inhibition pathway(e.g., by defective production and activation of the transcriptionfactor NF_(κ)B, as in the NOD mouse). Further, it is well known thatthere are two different TNF-alpha receptors. Defective signaling througheither receptor could render autoimmune cells susceptible to TNF-alphainduced apoptosis. As but another example, defective cell signalingthrough surface receptors that stimulate pathways that interact with thecell death pathway, i.e., LPS, IL-1, TPA, UV light etc., could renderautoimmune cells susceptible to apoptosis according to the theory of thepresent invention. Therefore, methods of the present invention that arebeneficial in the treatment of autoimmune disease are applicable to anyautoimmune patient that has a defect in a cell death pathway.

As mentioned above, current therapies for autoimmune disease aredirected toward decreasing the inflammatory reaction that is thought tobe responsible for destruction of self. TNF-alpha is part of theinflammatory response. Thus, according to the present theory, inductionof an inflammatory response, rather than inhibition of an inflammatoryresponse, is the preferred method of treating an autoimmune individual.This theory runs counter to existing dogma surrounding autoimmunetherapy today.

It is possible that TNF-alpha is inducing a cytokine, toxoid, or otherrelated molecule induced in the inflammatory response that is theresponsible for the benefit of TNF-alpha treatment. If so, induction ofinflammation via TNF-alpha treatment is still in agreement with thetheory of the invention. In a preferred embodiment, induction ofinflammation via TNF-alpha treatment induces mediators of autoimmunecell death.

A Novel Assay for Monitoring Treatment

It is well known that prolonged TNF-alpha treatment by itself is highlytoxic. In light of the elucidation of the cell death pathway describedabove, we hypothesized that the knowledge of this pathway could enabledevelopment of a sensitive in vitro assay that could be used to monitorthe in vivo effect of a particular TNF-alpha treatment regimen (i.e.,any treatment regimen that results in induction of TNF-alpha andinflammation). More particularly, a monitoring system could be developedthat combined the administration of TNF-alpha alone with an assaycapable of measuring the effect of TNF-alpha treatment on apoptosis ofautoimmune cells in a mammal diagnosed with an autoimmune disease. Sucha monitoring system would make it possible to measure the effect ofparticular doses of TNF-alpha on the apoptosis of autoimmune cellsconcurrently with treatment of an autoimmune individual. Moreover, sucha monitoring system would enable optimization or adjustment of the doseof TNF-alpha (i.e., or TNF-alpha inducing agent) to maximize autoimmunecell death, while minimizing exposure of the mammal to toxic doses ofTNF-alpha.

Thus, the invention provides a method of increasing or maintaining thenumber of functional cells of a predetermined type in a mammal thatinvolves a) treating a mammal to kill or inactivate autoimmune cells ofthe mammal; b) periodically monitoring the cell death rate of theautoimmune cells (i.e., by assaying the cell death rate of autoimmunecells in the mammal, wherein an increase in cell death rate of autoreactive T-lymphocytes indicates an increase in the number of functionalcells of the predetermined type (i.e., resistant to cell death)); and(c) periodically adjusting the dosage of the agent based on theinformation obtained in step (b). The autoimmune cells of the presentinvention include any cell defective in protection from apoptotic celldeath by any stimulus, for example, TNF-alpha, CD40, CD40L, CD28, IL1,Fas, FasL, etc.

The assay of step (b) allows one to identify novel formulations ofTNF-alpha, TNF-alpha inducing agents, TNF-alpha agonists, or agents thatact on the TNF-alpha signaling pathway effective in inducing apoptosisof T-lymphocytes or antigen presenting cells, that can be administeredover a longer course of treatment than was possible prior to the presentinvention (e.g., preferably over a period of months, more preferablyover a period of years, most preferably over a lifetime).

In a related embodiment, the present monitoring system may be used toidentify new doses, durations of treatment, and treatment regimens forTNF-inducing agents that were previously discounted as useful treatmentsbecause there was no way to monitor their effect. For example, incontrast to a preliminary report identifying BCG, a TNF-alpha inducingagent, as a useful type I diabetes treatment (Shehadeh et al., Lancet,(1994) 343:706), researchers failed to identify a therapeutic dose ofBCG because there was no way to monitor the effect of BCG in vivo (Allenet al., Diabetes Care (1999) 22:1703; Graves et al., Diabetes Care(1999) 22:1694).

The assay of step (b) may also be used to tailor TNF-alpha inductiontherapy to the needs of a particular individual. For example, asmentioned above, in one preferred embodiment, the assay of step (b) canbe carried out every day or every other day in order to measure theeffect of TNF-induction therapy and/or cell death inducing agents onautoimmune cell death rate so that adjustment to the administered dose,duration of treatment (i.e., the period of time over which the patientwill receive the treatment), or treatment regimen (i.e., how many timesthe treatment will be administered to the patient) of TNF-alpha can bemade to optimize the effect of TNF-alpha treatment and minimize theexposure of the patient to TNF-alpha or other cell death inducingagents. Of course, the skilled artisan will appreciate that the assaycan be performed at any time deemed necessary to assess the effect of aparticular regimen of TNF-alpha induction therapy on a particularindividual (i.e., during remission of disease or in a pre-autoimmuneindividual).

The assay can be used to tailor a particular TNF-alpha induction regimento any given autoimmune disease. For example, the in vitro monitoring ofselective killing of autoimmune cells can be used to selectively gradethe drug (i.e., adjust the dose administered to maximize the therapeuticeffect). The monitoring system described herein can be used to monitorin vivo trials of TNF-alpha treatment by continuously measuring theelimination of autoimmune cells, e.g., autoreactive T lymphocytes, withcontinuing sensitivity. Of course, the skilled artisan will appreciatethat the present monitoring system can be used to measure the effect ofTNF-alpha on in vivo killing of autoimmune cells in cases whereTNF-alpha-induction therapy is cited in conjunction with any othertherapy, e.g., T cell re-education, as described herein.

It is well known that TNF-alpha induction therapy has been shown to beineffective in patients with established autoimmunity, e.g., establisheddiabetes, but is effective in patients in a pre-autoimmune state, e.g.,patients in a pre-diabetic or pre-lupus state. In addition, it has beenestablished that TNF-alpha induction in adult NOD and NZB mice (a murinestrain susceptible to lupus-like disease) decreases diabetic or lupussymptoms respectively. According to the invention, TNF-alpha therapy canbe effective even in patients with established disease, by monitoringthe elimination of autoimmune cells and optimizing the dose, duration oftreatment, and/or re-treatment schedule accordingly. Thus, the assay ofstep (b) may be used to identify an effective dose, duration oftreatment, or treatment regimen of TNF-alpha (e.g., lower than dosespreviously shown to be ineffective in treatment of diabetes,particularly in the treatment of established diabetes) that can be usedas an effective treatment for autoimmune disease.

In another preferred embodiment, the assay of step (b) is used toidentify a dose, duration of treatment, or treatment regimen ofTNF-alpha that can reduce or eliminate side effects associated with aparticular autoimmune disease. A particular dose of TNF-alpha may beidentified that reduces or eliminates the symptoms associated with, forexample, vascular collapse associated with diabetes, blindness or kidneyfailure associated with Type 1 diabetes, or skin eruptions associatedwith lupus. It is well established that it is the side effectsassociated with the autoimmune reaction that are often responsible formortality of autoimmune patients. Thus, in one preferred embodiment, themonitoring system of the present invention identifies a treatmentregimen for TNF-alpha that reduces the symptoms and/or complications ofthe autoimmune disease, such that the quality of life of the patient isimproved and/or the life-span of the patient being treated is prolonged.In a related embodiment, the monitoring system of the present inventionidentifies a treatment regimen for TNF-alpha that prevents diseaseprogression or even halts disease in a patient diagnosed with anautoimmune disease.

Thus, in another aspect, the present invention provides a monitoringsystem for measuring the rate of cell death in an autoimmune mammal,including (a) a treatment regimen for killing or inactivating autoimmunecells in a mammal; and (b) an assay capable of measuring the effect ofthe treatment regimen on the cell death rate of autoimmune cells in themammal, wherein an increase in cell death rate indicates an decrease inautoimmunity.

In Vitro Assay for Monitoring Cell Death

The present invention provides a novel assay for monitoring apoptosis ofautoimmune cells in a mammal. In one preferred embodiment, the presentinvention provides an assay involving (a) isolating a blood sample froma mammal, preferably a human, and (b) testing the blood sample in vitrofor killing of autoimmune cells compared to non-autoimmune cells usingtechniques available in the art. As mentioned above, non-autoimmunecells are generally resistant to TNF-alpha induced apoptosis. Anincrease in cell death in autoimmune cells compared to non-autoimmunecells indicates that the dose of TNF-alpha or other cell death inducingagent is sufficient to induce killing of the autoimmune cells ordefective bone marrow origin cells.

Combined TNF Induction Therapy

The present invention also features a drug combination that includes twoor more TNF-alpha inducing agents. One particularly preferred combinedTNF-alpha treatment is the combination of TNF-alpha and IL1. Thistreatment strategy goes against the current dogma surrounding treatmentof autoimmune disease. For example, at the TNF Second InternationalMeeting (A Validated Target with Multiple Therapeutic Potential, Feb.24-25, 1999, Princeton, N.J., USA) it was disclosed that a combinationof anti-TNF-alpha antibody and anti-IL1 would be advantageous in thetreatment of autoimmune disease. The treatment of the present inventiondiscloses induction of inflammation, which is the opposite of thetreatment believed to be effective by those skilled in the art, that is,suppression of inflammation. Of course, in the current treatment,inflammation does not occur because the inflammatory cells actually dieprior to arriving at the target site or are killed at the target site.

Of course, the present invention is not limited to a combined TNFinducing therapy that includes only the combination of TNF-alpha andIL1, but includes any combination of TNF-alpha-including therapies,e.g., vaccination with BCG etc., viral infection, LPS, activation ofcells that normally produce TNF-alpha (i.e., macrophages, B cells, and Tcells), the chemotactic peptide fMet-Leu-Phe, bacterial and viralproteins that activate NF_(κ)B, agents that induce signaling pathwaysinvolved in adaptive immune responses (i.e., antigen receptors on B andT cells, CD28 on T cells, CD40 on B cells), agents that stimulatespecific autoreactive cell death receptors (i.e., TNF, Fas (CD95), CD40,p75NF p75TNF, and lymphotoxin Beta-receptor (LtbetaR), drugs thatstimulate TNF-alpha converting enzyme (TACE) which cleaves the TNF-alphaprecursor (i.e., to provide biological activity capable of stimulatingenhanced production or enhanced cytokine life after secretion) etc.

Identification of Inflammation-inducing Agents

In preferred embodiments, the present invention provides inflammatoryagents for the treatment of autoimmune disease that are counter to theanti-inflammatories used to treat autoimmune diseases today. Forexample, current methods for treating autoimmune disease includeTNF-alpha antagonists. Thus, the present invention provides TNF-alphaagonists (i.e., chemicals, peptides, or antibodies) that act on aTNF-alpha receptor. Other preferred treatments could fall under thecategories of drugs that act in opposite to anti-TNF-alpha agonists,anti-TNF-alpha antibodies, TNFR2 fusion proteins (Immunex), Embrel,anti-IL1 therapies, TNF-alpha convertase inhibitors, p38 MAP kinaseinhibitors, phoshodiesterase inhibitors, thalidomide analogs, andadenosine receptor agonists.

In another preferred embodiment, the invention allows for theidentification of drugs that induce cell death or selectively hamper theautoimmune cells by binding to cell surface receptors or interactingwith intracellular proteins. For example, drugs that stimulate the IL-1pathway or drugs that interact with converging pathways such as Fas,FasL, TACI, ATAR, RANK, DR5, DR4, DCR2, DCR1, DR3, etc. The drugs of thepresent invention can be characterized in that they only kill autoimmunecells having a selective defect in a cell death pathway which can becharacterized by two distinct phenotypes, (1) defects in antigenpresentation for T cell education and (2) susceptibility to apoptosis.

It will be appreciated that the above-described assay for monitoringdeath of autoimmune cells can be used to identify novel TNF-alphainducing agents and other inflammatory agents useful in the presentinvention. In preferred embodiments, autoimmune cells (i.e., anautoimmune cell isolated from a mammal diagnosed with autoimmunedisease) are exposed to a putative inflammatory or TNF-alpha inducingagent and assayed for increased cell death, an increase in cell death ofautoimmune cells compared to non-immune cells indicating identificationof a drug according to the present invention. Furthermore, autoimmuneblood could be exposed to chemical libraries for preferred and selectivecell death of yet unknown targets compared to non-autoimmune cells. Awide variety of chemical libraries are available in the art and can bescreened by use of the assay of the invention, which measures the rateof apoptosis of autoimmune cells.

In a related aspect, the above-described assay for monitoring death ofautoimmune cells can be used to identify autoimmune cells having the twodistinct phenotypes described above. In contrast to typical geneticapproaches for identifying cells carrying genetic defects, sensitivityto cell death may serve as the initial identification marker. Oncecell-death sensitive cells are identified, they can be assessed as towhether they also have the class I antigen presentation defect. Thus,the present invention provides a method of identifying autoimmune cellsby (1) assaying the cells for a susceptibility to apoptosis and (2)assaying for defects in antigen presentation required for T celleducation.

A Novel Combination Therapy

The data presented in Table 1 and described in detail in Example 1,below, demonstrate the remarkable success of combining two methods toinduce long-term normoglycemia with islet allograft transplantation inan already diabetic NOD host. The invention combines two therapies aimedat two separate targets of the immune system. The invention tests thisconcept by combining my prior transplantation technology with anautoimmune strategy to thwart the underlying disease, and for the firsttime provides long-term normoglycemia in naturally diabetic hosts viatransplantation with allogeneic islets. Thus, the invention, views therejection problem as one involving two immune barriers, i.e., the graftrejection barrier and the recurrent autoimmunity barrier. To address thegraft rejection barrier, I used donor antigen modified islets, and forthe recurrent autoimmune barrier I used CFA, a strong inducer ofTNF-alpha.

TABLE 1 Host Donor Treat- Individual Survival (days) Groups StrainStrain ment Days of normoglycemia Mean 1. C57BL/6 NOD- — 2, 2, 3, 9, 237.8 IDDM* 2. β₂M^(−/−) NOD- — 5, 9, 12, 12, 17, 18, 71 20. IDDM 3.C57BL/6 BALB/C — 5, 5, 7, 10 6.7 4. β₂M^(−/−) BALB/C— >100, >100, >100, >100 >100 5. NOD NOD- — 5, 10, 12, 13 10 IDDM 6. NODNOD- CFA 12, 26, 30, >38, >66, >59 IDDM >120, >122 7. C57BL/6 NOD- CFA10, 10, 10 10 IDDM 8. β₂M^(−/−) NOD- CFA 5, 14, 32, 36, >59, >79, >57IDDM >115, >115 *IDDM stands for insulin-dependent diabetes melitus.

Table 1, above, represents a series of experiments that were carried outin which host mice were treated to prevent recurrent autoimmunity, viakilling or inactivation of autoreactive lymphocytes, and thentransplanted with donor islet cells in which rejection triggeringantigens had been eliminated or modified.

The mice were injected once intraperitone ally with complete Freund'sadjuvant (CFA) (25 μl/mouse) to induce TNF-alpha.

The same day, islet cell transplantation was carried out as follows. Thedonor antigen modified islet cells were isolated from transgenic β₂M (β2microglobulin) knockout mice purchased from the Jackson Labs. Asmentioned above, the β₂M gene encodes a critical chaperone proteinessential for surface expression of class I proteins. Host β₂M, a highlyconserved protein, can in part re-constitute β₂M.

Transgenic or normal islet cells were transplanted into nine groups ofmice. Three of the groups (groups 6, 7, and 8) were pre-treated withCFA; the other six groups were not pre-treated.

As is shown in Table 1, naturally diabetic mice (NOD-IDDM) that receivedtransgenic transplants but were not pre-treated with CFA (group 2) hadmean survival times of 20 days, suggesting that the protection of donortissue from graft rejection does not protect the tissue from anestablished autoimmunity. Likewise, group 7 establishes that hosttreatment with CFA, an immunomodulator now believed to modifyexclusively the autoimmune response, does not protect normal allogeneicdonor cells from rapid graft rejection. In contrast, the CFA treateddiabetic mice receiving transgenic transplants (groups 6 and 8) survivedover 57 days (mean). The remaining groups were additional controls:group 1 (no CFA; diabetic host, non-transgenic donor cells); group 3 (noCFA; non-diabetic host, non-transgenic donor cells); group 4 (no CFA;non-diabetic host; non-transgenic donor cells); and group 7 (CFA;diabetic host; non-transgenic donor strain). As is shown in Table 1, theonly one of these control groups exhibiting longevity were non-diabetichosts receiving transgenic donor cells, a therapy known to thwart graftrejection (group 4).

At approximately 120-130 days post transplantation, the transplantedsyngeneic and allogeneic islets were removed by nephrectomy. This was acontrol experiment to prove the animals reverted back to hyperglycemia.

The NOD mice receiving the syngeneic transplant had, within 24 hrs,blood sugars in excess of 500 mg/dl and needed to be sacrificedimmediately because of their severe diabetic state. Histology on thesemice showed that the transplanted islets in the kidney survived in somecases but did not appear, in all cases, healthy. There were granulatedislets, but massive lymphocytic infiltrates surrounded and invaded theislet tissue. The islet invasion by host lymphocytes is a histologictrait indicative of autoreactivity against the islet tissue. Theendogenous pancreas demonstrated no surviving islets and was dotted withlarge lymphocytic clusters, presumably at sites of former islet tissue.

The NOD mice receiving the allogeneic islets, in contrast, remainednormoglycemic after the nephrectomies had been performed to remove theallogeneic islet tissue. No change in blood sugar was noted. Afterapproximately seven days of this perfect blood sugar control, the micewere sacrificed. Histologic examination showed that endogenous islets inthe pancreas were regenerated. The islet number was less than normal,but the islets present were large, healthy, and had no lymphocyteinvasion (although they did have a characteristic NOD rim of lymphocytessurrounding the healthy islet). In contrast, the allogeneic grafts weregone in most cases by this late 120-140 day post-transplantation timepoint. These results support the thesis that what occurred was rescueand regeneration of the endogenous pancreas. The results support thatthe immune system was additionally re-educated.

Thus, in one preferred embodiment, the invention provides a method ofinhibiting rejection of transplanted islet cells in a diabetic patient,by (a) pre-treating the islet cells to modify, eliminate, or mask isletcell antigen otherwise capable of causing T-lymphocyte-mediatedrejection response in a patient, together with (b) treating the patient(prior to, during, or following transplantation) to kill or inactivateautoreactive host lymphocytes that are otherwise capable of killing ordamaging the transplanted islet cell.

In preferred embodiments, step (a) involves genetically altering thedonor animal so that HLA class I or a molecule in its pathway isgenetically deleted or chaperone ablated to prevent surface expression,or masking HLA class I antigen using an antibody F (ab′)₂ fragment thatforms a complex with HLA class 1; and step (b) involves administering toa patient TNF-alpha, or a TNF-alpha inducing substance, e.g., tissueplasminogen activator (TPA), LPS, IL-1, TV light, such as aninteracellular mediator of the TNF-alpha signaling pathway or an inducerof cell death in defective cells.

Class I Antigen Presentation

Prior to the experiments described above, I observed that a small amountof class I ablation was beneficial for the inhibition of rejection ofdonor islets in diabetic NOD mice (Faustman et al., Science (1991)252(5013):1700-2). Based on these results, I proposed that a morecomplete and permanent class I ablation might even be better for longterm graft survival. To achieve a more permanent class I ablation, Itransplanted F2 islets that were ablated for both the β₂M (β2microglobulin) gene and Tap 1 into already diabetic NOD mice (MHC classI^(−/−,−−)) (see Example 3). The β₂M gene encodes a critical chaperoneprotein essential for surface expression of class I peptides. The Tap 1gene encodes a protein required for transport of endogenousself-peptides into the endoplasmic reticulum for stable peptide andclass I assembly before presentation on the cell surface. Surprisingly,only one of the six mice exhibited long term graft survival. Individualgraft survival times (days) for the six mice were: 11, 12, 13, 14, 14,and 71. These unexpected results suggested that the reexpression ofpeptide and class I was a step that was not only not harmful, but wasactually necessary for immune system re-education leading to endogenousislet regeneration and rescue. Thus, in the present application, Ipropose, without limiting the biochemical mechanism of the invention,that some intact MHC class I molecules are required for the re-educationprocess to occur.

Based on the above-described results, it appears that the graftrejection barrier actually serves two important functions that appear tocontribute to successful islet cell regeneration in this model.Temporary class I ablation (class I^(−/−)) serves initially to protectthe graft from immediate rejection. Subsequently, MHC class I proteinsare reexpressed and exchanged on the graft by 24-72 hourspost-transplantation through abundant host β₂M proteins from the serum(Anderson et al., J. Immunol. (1975) 114(3):997-1000; Hyafil et al.,Proc. Natl. Acad. Sci., USA (1979) 76(11):5834-8; Schmidt et al.,Immunogenetics (1981) 13:483-91; Bernabeu et al., Nature (1984)308(5960):642-5; Li et al., Transplantation (1993) 55(4):940-6).Surprisingly, subsequent reexpression of endogenous peptide via MHCclass I appears to contribute to the reeducation of T lymphocytes withproper negative selection of autoreactive cells. In further support ofthis observation, I demonstrated, that to be therapeutically effective,at least one MHC class I K or D locus of the donor lymphocytes needed tobe matched to the corresponding locus in the NOD host mouse (i.e.,either the K^(d) or D^(b) locus should be present on the donor MHC classI expressing cells). Therefore, the reeducation component of thecombination therapy requires that the MHC class I expressing cells,presenting the self-peptide, be semisyngeneic or fully syngeneic to theautoreactive cells in the NOD host mouse. Coupled with the selectiveelimination of autoreactive lymphoid cells by treatment with CFA, thepresent combination therapy provides a powerful treatment for autoimmunedisease where regeneration of tissue is desired.

Maintenance of Transplanted Islet Cells is not Required for Regenerationof Endogenous Pancreas

The experiments described above further suggested to me that maintenanceof the transplanted islets in vivo might not be necessary for endogenouspancreatic islet cell regeneration. In order to test this theory,transgenic MHC class I^(−/−) islet grafts were transplanted into alreadydiabetic NOD mice with TNF-alpha induction. Transgenic MHC class I^(−/−)islet grafts placed under the kidney of diabetic NOD mice were laterremoved by nephrectomy at various times post-transplantation. Asdescribed in Example 2, all mice remained normoglycemic for at least 120days after nephrectomy and the pancreatic histology revealed beautifulendogenous pancreatic islet regeneration. In contrast, NOD mice thatreceived syngeneic islet transplants rapidly returned to hyperglycemiapost-nephrectomy.

As proposed above, these data support the theory that for endogenousregeneration of islets, or other regenerating tissue subject to immuneattack (e.g., hepatic cells), maintenance of the transplanted isletcells is not essential to endogenous pancreatic islet cell regenerationand rescue. Thus, the invention, in one respect, views the problem oftissue regeneration and rescue in autoimmunity as one involving twodifferent barriers, (i.e., the recurrent autoimmunity barrier and there-education barrier). The required steps for tissue regeneration appearto be: (1) ablate the host autoimmune cells (e.g., by killing orinactivation); and (2) re-educate the immune system with class I andpeptide to protect the regenerating pancreas.

Thus, the present invention provides a method of reestablishing systemictolerance and eliminating existing autoimmunity that promotesregeneration and rescue of cells and tissue.

Treatment by Injection of MHC Class I and Peptide

Based on the discovery that class I peptide presentation is required forre-educating the NOD host and the knowledge that maintenance oftransplanted islet cells is not required for endogenous islet cellregeneration, I proposed that islet transplantation and isolation mightnot be necessary for in situ islet regeneration in setting ofautoimmunity. Islet isolation and transplantation are laboriousprocedures with associated supply and demand limitations in the clinicalsetting. A procedure for increasing the number of functional islet cellsthat does not require islet isolation and transplantation would providegreat benefit to the treatment of diabetes. This method of treatmentcould be extended to other autoimmune diseases where immune reeducationis desired (U.S. Pat. No. 5,538,854).

I proposed that mere injection of functional cells expressing class I(class I⁺), or even MHC class I/peptide complex, into a mammal, withconcurrent ablation of autoimmune cells, would be efficacious intreating a diverse array of autoimmune diseases. For example, normalpancreatic islets express MHC class I and have few associated passengerlymphocytes that express both MHC class I and class II molecules (thispreparation is referred to herein as B6 splenocytes). In the case ofdiabetes, a preparation of normal pancreatic islet cells may be injectedinto a patient to achieve exposure to class I antigen. Although donorcell survival may be short lived, repeated exposure might be sufficientto re-educate the host immune system with concurrent ablation ofautoimmune cells. In cases where donor cell preparation is tedious orpoor donor cell survival time is limiting the efficacy of the method,class I/peptide complex may be administered directly to the host.

In order to test this hypotheses, diabetic NOD mice were initiated on a40 day regimen of one bolus injection of CFA to transiently induceTNF-alpha and biweekly exposure by intravenous injection to B6splenocytes (class I⁺) (Example 4). As predicted, the injectedsplenocytes survived only transiently in the host due to rejection.However, transient elimination of autoimmune cells (e.g., viaCFA-mediated TNF-alpha induction) combined with repeat exposure to B6MHC class I and peptide was sufficient for reversal of diabetes inapproximately 30% of diabetic NOD hosts. Partial protection was achievedin approximately 50% of the diabetic NOD hosts, but the percentage ofhost NOD mice remaining normoglycemic increased to 80% after allowingthe islets to regenerate for more than 120 days. Furthermore, since thedonor B6 splenocytes have a MHC class I phenotype of K^(b)D^(b) and thehost NOD mice have a MHC class I phenotype of K^(d)D^(b), I was able todetermine that the now normoglycemic host NOD mice had insulin secretingpancreatic cells of donor B6 origin, based on K^(b) antibody staining.In addition, to support this finding, I determined that only injectionof live, non-irradiated, splenocytes resulted in a contribution of thedonor MHC class I phenotype to the host insulin secreting cells.

Blood sugar levels were poorly controlled in mice receiving theinjection therapy described above. Fluctuations in blood sugar levelcould negatively influence benefit of the combined injection therapy. Inorder to control for this variant, additional groups of diabetic NODmice were similarly treated with TNF-alpha induction and B6 splenocyteinjection, but with simultaneous intraperitoneal implantation of B6islets encapsulated with alginate (referred to herein as a euglycemicclamp). A euglycemic clamp provides a membrane barrier system thatallows short term glycemic control of insulin exchange but preventsdirect cell-cell contact (e.g., for T cell education). After 40 days,the encapsulated islets were surgically removed and blood sugar levelsof the diabetic NOD mice were monitored for evidence of in situ pancreasregeneration. Remarkably, diabetic NOD mice that had received biweeklyB6 splenocyte immunizations and a single dose of TNF-alpha inductiontherapy remained normoglycemic for 40 days after clamp removal in 78% ofthe cases. Moreover, after the therapy was halted and autoimmunityeliminated, the continuous expansion of the endogenous pancreas wassufficient for sustained tolerance to self antigens. In contrast, incontrol experiments, where splenocytes permanently ablated for MHC classI proteins (MHC class I^(−/−,−/−)), poor in situ islet regeneration wasobserved (Table 3, group 4, FIG. 5). However, injection of splenocyteslacking MHC class II proteins (MHC class II^(−/−)) permitted in situislet regeneration, presumably due to continued expression of endogenouspeptide in the context of MHC class I (Table 3, group 5, FIG. 5).Therefore reestablishment of self tolerance and elimination ofautoreactivity was MHC class I dependent.

Therefore, I have identified and optimized a novel combination treatmentfor diabetes melitus mellitus. Thus, in yet another aspect, the presentinvention features a method of increasing and preserving the number offunctional cells of a predetermined type in a diabetic patient thatincludes the steps of (1) ablation of autoimmune cells, (2) exposure toMHC class I and peptide, and (3) maintenance of glucose control. Asmentioned above, exposure may occur, for example, either bytransplantation of functional MHC class I and peptide presenting cellsof a predetermined type, or preferably by repeated injection of suchcells. Alternatively, exposure to MHC class I and peptide may occur byinjection of class I/peptide complexes, peptide feeding of autologouscells etc.

In a particularly preferred embodiment, the present invention provides amethod of increasing the number of functional cells of a predeterminedtype in a diabetic patient that includes the steps of (1) ablation ofautoimmune cells (i.e., cells that are defective in cell death), (2)exposure to MHC class I and peptide by repeated injection of functionalcells of a predetermined type, expressing peptide in the context of MHCclass I (or MHC class I/peptide complex), and (3) maintenance of glucosecontrol. In the case of diabetes, the functional cells of apredetermined type include islet cells, for example, B6 splenocytes.Maintenance of blood glucose levels may be achieve by any means known inthe art, for example, insulin injection, or by use of a euglycemicclamp. The diabetic patient can be any mammal, preferably a humanpatient.

Treatment of Autoimmune Disease

Based on the discoveries described herein, I have devised a noveltherapy for the correction of any established autoimmunity. Used incombination, exposure to self peptide in the context of MHC class I andkilling or inactivation of autoreactive lymphocyte permits theendogenous regenerative potential of mammalian tissue to be enacted. Inaddition, the present treatment enables preservation and rescue ofexisting tissue. The effect of this combination therapy is there-education of the immune system with the simultaneous reversal ofautoimmunity within the host.

With respect to diabetes treatment, I further hypothesize thatsuccessfully regenerated pancreatic B6 islet cells that hyper-expressMHC class I and peptide (e.g., determined by histological examination)maintain peripheral tolerance once sufficient islet growth has beenestablished. In vivo exposure to MHC class I and peptide expressingcells by transplantation or injection appears to initiate theeducational process for long-term and stable tolerance, beyond theperiod of treatment.

Several striking similarities exist between the NOD mouse and humandiabetic patients, suggesting that this novel therapeutic approach canbe easily applied to treat human diabetic patients. For example,diabetic human splenocytes, like murine splenocytes, have defects inresistance to TNF-alpha induced apoptosis (Hayashi et al., supra). Inaddition, like NOD mice, human splenocytes have age related defects inMHC class I presentation of self peptides for proper T cell selection(Faustman et al., supra; Fu et al., J. Clin. Invest. (1993) 91:2301-7).Finally, it has been recognized for years that even after a severehyperglycemic episode, diabetic humans continue to produceautoantibodies to islet targets, indicating that the islet cells orislet precursor cells of the pancreas were not completely ablated. Thisindicates that humans diagnosed with diabetic autoimmunity may have highislet regenerative potential.

Thus in one aspect, the invention features a method of increasing thenumber of functional cells of a predetermined type in an individualdiagnosed with an autoimmune disease, by (1) providing a sample offunctional cells expressing MHC class I and peptide, (b) exposing amammal to the MHC class I and peptide, and (c) prior to, after orconcurrently with step (b), treating the mammal to kill or inactivateautoimmune cells (i.e., cells defective in apoptosis) in the mammal.

Where the mammal is a diabetic human patient, it may be desirable to adda further step of maintaining normal levels of glucose prior to, after,or concurrently with step (b). As described above, maintenance of normalblood glucose levels in a patient with established diabetes may improvethe efficacy of the inventive method.

As mentioned previously, re-education of the immune system with MHCclass I and peptide can employ cells expressing endogenous peptide inthe context of MHC class I or class I /peptide complexes alone. A numberof such immune system re-education methods are known, e.g., as describedin U.S. Pat. No. 5,538,854, hereby incorporated by reference.

Similarly, a variety of well known methods can be used in the presentinvention to accomplish ablation of autoimmune cells. One preferredtreatment is the administration of TNF-alpha, which is available fromGenentech Corporation, South San Francisco, Calif.; Roche; BoehringerIngelheim; Asahi Chemical Industry; and Sigma Chemicals. Theadministration intraperitoneally of TNF-alpha to decrease rejection indiabetes-prone mice is described in Rabinovitch et al., J. Autoimmunity(1995) 8(3):357-366, hereby incorporated by reference.

Other host treatment methods can be used as well to ablate autoimmunecells, for example, administration of CFA, interleukin-1 (IL-1),proteasome inhibitors, NF_(κ)B inhibitors, anti-inflammatory drugs,tissue plasminogen activator (TPA), lipopolysaccharide, UV light, or anintracellular mediator of the TNF-alpha signaling pathway. Such agentsinduce the apoptosis of autoreactive lymphocytes by interrupting thepathway downstream from TNF-alpha receptor signaling. Other usefulagents are drugs that act downstream of TNF-alpha receptor binding.(Baldwin et al., Ann. Rev. Immunol.(1996) 12:141; Baltimore, Cell (1996)87:13).

In other aspects, the invention features a method of increasing thenumber of functional cells of a predetermined type in an individualdiagnosed with an autoimmune disease, by (a) providing a sample of cellsof the predetermined type, (b) treating the cells to modify thepresentation of an antigen of the cells that is capable of causing an invivo T-lymphocyte-mediated rejection response, (c) introducing thetreated cells into the mammal, and (d) prior to, after, or concurrentlywith step (c), treating the mammal to kill or inactivate T-lymphocytesof the mammal. This method may be particularly useful for the treatmentof advanced-stage autoimmune disease, where complete destruction of aparticular cell type or tissue has been achieved.

In preferred embodiments, step (b) involves eliminating, reducing ormasking the antigen. A number of methods can be used to modify,eliminate, or mask donor cell antigens; some of these are described inthe afore-mentioned Faustman U.S. Pat. No. 5,283,058. For example, step(b) may involve genetically altering the donor animal so that HLA classI or a molecule in its pathway is genetically deleted or chaperoneablated to prevent surface expression. Alternatively, step (b) mayinvolve masking HLA class I antigen using an antibody F (ab′)₂ fragmentthat forms a complex with HLA class I.

The therapeutic regimen of the present invention can be used not just toinhibit rejection of regenerating cells, but also to treat autoimmunediseases in which endogenous cell or tissue regeneration is desired,e.g., to allow myelin regeneration (or mere preservation of theremaining autoimmune target cells that are surviving) in multiplesclerosis or joint regeneration in rheumatoid arthritis.

Where the invention is used not just to protect regenerating endogenouscells, e.g., islet cells, from autoimmune attack, but also to protecttransplanted cells and tissues, the methods described above can becombined with other, known methods for inhibiting allograft rejection.Such methods include administration of anti-alpha CD3 antibodies,anti-CD40L antibodies (CD40 Ligand, a co-receptor for T cell triggering,to prevent reduction of tolerance in the host), FK506, tacrolimus,sirolimus, alpha-CD25 induction, etc. and cyclosporin A. As is discussedabove, autoimmune insulin-dependent diabetes melitus mellitus (IDDM)lymphocytes are particularly sensitive to cell death via the TNF-alphapathway, and thus drugs that potentiate this pathway downstream ofreceptor binding can be employed. Examples of such potentiating drugsare targets of TRIP, NIK, IKK, TRADD, JUN, NF_(κ)B, Traf2, andproteasome processing etc.

Even when the primary goal is regeneration or rescue of endogenous cellsrather than permanent allograft engraftment, it can be useful to implantan allograft and promote its temporary survival, while simultaneouslypromoting re-education of the immune system so that the endogenous cellscan regenerate; autoreactive lymphocytes are detrimental to both theallograft and the regenerating cells, and therefore killing orinactivation of those cells is doubly advantageous.

Thus, in the case of diabetes, for example, transplanted islets can betemporarily protected from rejection by temporary encapsulation or bymeticulous blood sugar control with exogenous insulin, while the host istreated, as described above, to kill autoreactive lymphocytes and theimmune system is re-educated by methods using class I and peptide orclass II and peptide. An additional advantage of using allogeneic islettransplants during this phase is that normal islets that are temporarilyprotected might provide normal hormonal and secretory capacities whichwill optimize in situ regeneration and rescue.

Other Embodiments

The skilled artisan will appreciate that the present invention caneasily be applied to treat any of a variety of autoimmune disorders.Particularly, the present invention is particularly preferred for thetreatment autoimmunity where destruction of a particular cell type ortissue is ongoing. The present invention provides the advantage ofproviding relief to patients with even established cases ofautoimmunity, where tissue destruction is advanced or complete. Thepresent invention will now be demonstrated by the following non-limitingexamples.

EXAMPLES Example 1 Combination Therapy

To devise a clinically applicable protocol for the regeneration ofislets in a diabetic host, two therapies were combined and tested in thediabetic NOD mouse (a murine model for human type I diabetes). First,donor B6 islets were protected from graft rejection by temporary class Iablation (class^(−/−)) of the β₂M gene (Anderson et al., supra; Hyafilet al., supra; Schmidt et al., supra; Bernabeu et al., supra; Li et al.,supra). The transgenic donor B6 islets were then removed from the donormouse and transplanted into the host NOD mouse. Subsequently, a singlefoot pad injection of CFA was simultaneously administered; a treatmentthat sustains levels of TNF-alpha for days (Sadelain et al., Diabetes,(1990) 39:583-589; McInerey et al., Diabetes, (1991) 40:715-725; Lapchaket al., Clin. Immunol. Immunopathol. (1992) 65(2):129-134).

The response of severely diabetic NOD female mice to treatments of donorB6 islets with or without transient MHC class I^(−/−) interruption andTNF-alpha induction are summarized in Table 2.

TABLE 2 Blood Sugar Control in Diabetic NOD Mice Receiving IsletTransplants TNF-alpha* Days of Group Donor Induction normoglycemia Mean± SD #1 NOD − 4, 6, 6, 8, 10   9 ± 2.3 #2 B6 − 2, 2, 3, 9, 9, 23   8 ±8.0 #3 B6-Class − 5, 9, 12, 12, 17, 18, 71 22 ± 24 I^(−/−) #4 NOD + 30,55, 61, 70, 72, 121, 85 ± 40 136, 137 #5 B6 + 9, 9, 9, 10, 11  10 ± 0.9#6^(†) B6-Class + 13, 14, 14, 15, 25, 32, 32, >62 ± 54  I^(−/−) 32,36, >133, >133 >131, >129, >132 #7^(††) B6-Class + 11, 12, 13, 14,14, >148 35 ± 55 I^(−/−),^(−/−) *TNF-alpha induction was accomplishedwith a single foot pad injection of CFA at the time of the transplant.^(†)B6-Class I^(−/−) donor islets represent islets with transient classI interruption due to ablation of the donor β₂-microglobulin gene (β₂M).^(††)B6-Class I^(−/−),^(−/−) donor islets with more permanent class Iablation were isolated from mice with both β₂M and Tap1 geneinterruption, two chaperone proteins essential for class I surfaceexpression.

All hosts were female diabetic NOD mice, typically greater than 20 weeksof age, with sustained blood sugar levels in excess of 400 mg/dl for atleast 7 days with the administration of insulin of 0.5 U/kg to preventdeath. This dose of insulin typically maintains blood sugar levels ofNOD mice diabetic in the normal range of 100-200 mg/dl. Eight to twelvehours prior to transplantation, insulin is stopped. All islettransplants are performed unilaterally under the kidney capsule tofacilitate post-transplant islet histology using standard techniques.

Typically, NOD islets isolated form 5-10 week old pre-diabetic femaleNOD mice are rapidly rejected when transplanted into severely diabeticNOD mice (Table 2, group 1). Similarly, B6 islets transplanted under thekidney capsule of diabetic NOD mice are also rapidly rejected with amean survival time of 8±8.0 (Table 2, Group 2). As published in theliterature, although donor islets with MHC class I^(−/−) ablationsurvive indefinitely in non-autoimmune hosts (Faustman, 1991, supra),the transient MHC class I ablation only permits a three fold increase inislet survival in the challenging diabetic NOD host. All diabetic NODhosts eventually reject the B6 class I^(−/−) A donor islets; meansurvival is extended to 22±24 days (Table 2, group 3). As shown in Table2, group 4, although TNF-alpha induction facilitates syngeneic islettransplantation in NOD hosts, this autoimmune directed therapy hasminimal effect of B6 islet survival. B6 islets transplanted intodiabetic NOD mice with TNF-alpha induction are uniformly rejected by day10 post-transplantation in all diabetic NOD recipients (Table 2, group5).

As shown in FIG. 1, B6 islets isolated from young NOD mice andtransplanted into a diabetic NOD mouse with TNF-alpha inductiondemonstrate severe lymphocytic infiltrates under the kidney capsule atthe islet transplantation site (see also, FIG. 2). At the same time,blood sugar levels have increased to what they were prior totransplantation. In addition, the endogenous pancreas shows no intactislets; the remaining isle structures in the pancreas are obscured bydense pockets of infiltrating lymphocytes. Similarly, B6 isletstransplanted into an NOD mouse treated with TNF-alpha induction arerejected; the histology is virtually indistinguishable; massivelypocytic infiltrates under the kidney capsule at the transplant sitewith the endogenous pancreas showing islet structures obliterated withlymphocyte invasion (FIG. 1B).

Importantly, combining MHC class I^(−/−) islet transplantation withTNF-alpha induction in NOD diabetic hosts was successful (Table 2, group6). Continuous and sustained normoglycemia was observed in 5 of the 14diabetic NOD hosts; normoglycemia continued beyond 129 days after islettransplantation in the previously diabetic NOD mice receiving thecombined treatment. The mean survival time for normoglycemia exceeded62±54 days. Long-term normoglycemic NOD mice were sacrificed after atleast 129 days of post-transplantation monitoring to evaluate thesubrenal capsule islet transplantation site and the endogenous pancreas.

Surprisingly, all 5 long-term normoglycemic NOD mice receiving B6 classI^(−/−) islets with TNF-alpha induction treatment histologicallydemonstrate no surviving islet grafts under the kidney capsule at 130days post-transplantation. The endogenous pancreas of these micedemonstrated significant islet regeneration (FIG. 3). Furthermore, theislets in the pancreas lacked lymphocyte invasion or, at most,occasionally demonstrated circumferential lymphocytes surrounding theregenerated islets. As the individual animal histology in Table 3summarizes, in situ pancreas regeneration was exclusively a trait ofdiabetic NOD mice treated with TNF-alpha in combination withtransplantation of donor islets having transient MHC class I^(−/−)interruption. These results demonstrate that the above combinationtherapy (administration of TNF-alpha and islet cells temporarily ablatedfor class I (class I^(−/−))) successfully eliminates existingautoimmunity in severely diabetic NOD mice and promotes regeneration ofthe endogenous pancreas.

In addition to the elimination of the existing autoimmunity, eliminationof TNF-alpha sensitive cells in NOD mice by combined treatment of CFAand C57 lymphocytes or islets was also observed. As the scientificliterature reports, incubation of spleen cells from non-autoimmuneC57BL/6 mice treated with TNF-alpha (10-20 ng/ml) had no effect on cellviability. In contrast, high numbers of splenocytes from female NOD micewere killed by apoptosis after 12 hours of low dose TNF-alpha exposure.TNF-alpha induced apoptosis in NOD spleen cells from mice 7-15 weeks ofage was confirmed using either trypan blue staining or flow cytometry(FIG. 7). Importantly, splenocytes from formerly diabetic NOD mice whichreceived the therapeutic combined treatment of CFA plus class I positivecells were found to have permanent elimination of TNF-alpha sensitivelymphocyte subpopulations (FIG. 7). In vitro treatment of splenocytesfrom successfully treated NOD mice with TNF-alpha resulted in little orno cell death beyond that seen in the absence of TNF-alpha.

I have also examined the time course of the appearance and theelimination of TNF-alpha sensitive subpopulations. In pre-diabetic NODmice, which progress to diabetes, splenocytes were found to have highsensitivity to TNF-alpha induced apoptosis beginning after approximately7 weeks of age. The timing of the onset of this sensitivity wascorrelated with a lineage/tissue specific decrease in LMP2 protein.Indeed, splenocytes from LMP2^(−/−) mice have TNF-alpha sensitivity atall ages. Yet, only the combined treatment of CFA and MHC class I donorcells permanently eliminated these TNF-alpha autoreactive cells inautoimmune NOD mice.

To further prove that the elimination of the TNF-alpha sensitivesub-population of splenocytes is a key feature of the combined treatmentof CFA and MHC class I donor cells, I conducted an adoptive transferexperiment. This was accomplished by testing whether diabetes, fromspontaneously diabetic NOD mice, can be transferred to healthyirradiated (790R) syngeneic young male NOD mice. In these experiments, Iobserved that spleen cells from diabetic NOD mice adaptively transferreddiabetes into healthy mice, typically within 20-30 days after aninjection of 2×10⁷ splenocytes (FIG. 8). Importantly, when thelymphocytes from diabetic mice were treated in vitro with TNF-alpha at20 ng/ml for 24 hours prior to transfer, diabetes induction was notobserved during the 33 days of observation (FIG. 8). Accordingly,treatment of splenocytes with TNF-alpha in vitro has the ability toselectively kill existing disease-causing cells. Although other cellpopulations may participate in disease expression, their action is notsufficient to enable expression of diabetes in the absence of theTNF-alpha sensitive population.

Furthermore, in these adoptive transfer experiments, all male NOD micereceiving untreated or TNF-alpha treated diabetic lymphocytes, if stillnormoglycemic, were killed after the onset of diabetes or at day 35.Male NOD mice receiving the untreated diabetic splenocytes becamehyperglycemic during the observation period and had their pancreaticislets obliterated by dense infiltrates. All male NOD mice receivingTNF-alpha treated lymphocytes remained normoglycemic, but had mild tomoderate invasive insulitis. This indicated that, while diabetestransfer was delayed, diabetes most likely would occur with lengthenedobservation times. Therefore, TNF-alpha treatment of diabeticsplenocytes delays rapid diabetes transfer but does not permanentlyeliminate T cells with the latent potential to cause disease in theautoimmune prone host.

Indeed, this experimental result is complementary to the pancreatichistology observed in the NOD mice exhibiting diabetes that were treatedwith CFA alone and shown in FIG. 1. Active autoimmunity defined byinvasive insulitis is never eliminated but is reduced in magnitude byCFA treatment alone. To eliminate autoimmunity permanently, otherinterventions, such as re-introduction of syngeneic MHC class Iexpressing cells, as described herein, are required.

In addition, NOD diabetic mice successfully treated with the combinationof CFA and MHC class I donor cells no longer have TNF-alpha sensitivelymphocyte subpopulations, indicating that this population ofdisease-causing cells has been eliminated and continues to bepermanently eliminated (FIG. 7). Therefore, not only does the brief invivo therapy with CFA and cells bearing semi-matched MHC class I peptidecomplexes eliminate existing autoimmune cells but also the combinedtherapy established permanent elimination of these disease causingcells. Splenocytes from a successfully treated long-term normoglycemicNOD mice do not transfer diabetes after 100 days of observation and thehost pancreatic histology revealed no lymphocytic infiltrates.

Example 2 Maintenance of Transplanted Islet Cells is not Required forRegeneration of the Endogenous Pancreas

To confirm the ability to eliminate existing autoimmunity and regeneratethe endogenous pancreas, additional diabetic NOD mice were transplantedwith MHC class I^(−/−) islets under the kidney capsule with TNF-alphainduction. At various times post-transplantation, in the presence ofsustained induced normoglycemia, islet grafts placed under the kidneywere removed by nephrectomy and survival surgery performed to evaluatewhether maintenance of normal blood sugar levels was dependent onpresence of the graft. FIG. 3 shows that all five severely diabetic micethat successfully received TNF-alpha induction and B6 MHC class I^(−/−)islet therapy remained normoglycemic at sacrifice times 3 to 60 daysafter nephrectomy. In addition, the pancreatic histology in all fivehosts revealed a surprising number of pancreatic islets, with minornumbers of circumferential lymphocytes or no lymphocytes surrounding theregenerated and rescued islets. Evaluation of all islet transplant sitesunder the kidney demonstrated no surviving transplanted islets.

In marked contrast, all NOD mice receiving syngeneic NOD islet cells bytransplantation, in conjunction with TNF-alpha induction, rapidlyreturned to hyperglycemia post-transplantation, demonstrating failure ofthis transplant protocol using syngeneic NOD islets to promoteendogenous pancreatic islet cell regeneration.

Example 3 Temporary Class I^(−/−) Ablation is Critical for SuccessfulCombination Therapy Treatment

In order to begin to dissect the mechanism of systemic reestablishmentof tolerance sufficient for pancreatic islet re-growth, additionalexperiments were performed. In order to achieve a higher success rate ofpancreatic islet regeneration with eliminated autoimmunity, a morepermanent MHC class I ablated islet was tested in the combinationtherapy treatment. Islets from B6 donors with both ablated β₂M and Tap 1genes (MHC class I^(−/−,−/−)), the obligatory chaperone and transportproteins for MHC assembly, respectively, were transplanted into severelydiabetic NOD mice with TNF-alpha induction. I found that although thisapproach is effective at prolonging normoglycemia in murine hostswithout autoimmunity, this treatment failed to prolong normoglycemia inthe autoimmune, diabetic NOD host. More permanent donor MHC class Ielimination with TNF-alpha induction in the diabetic NOD host culminatedin rapid islet graft rejection and poor ability to achieve endogenousislet regeneration (Table 2, group 7). Apparently, some expression ofdonor MHC class I and self peptide is essential for NOD toleranceinduction to self antigens, even if only for a limited time, on average20 days (Table 2, group 3) before transplant rejection.

Example 4 Injection of Temporarily Ablated Islet Cells is Sufficient forInduction of Endogenous Pancreas Regeneration

Based on the data of Example 3, above, I proposed that class I+lymphocyte immunizations could be an efficacious therapy, even if thedonor cells only survived a short time in vivo post-injection. In orderto test this theory, nine diabetic NOD mice with severe hyperglycemiawere initiated on a 40 day regimen of one bolus injection of CFA totransiently induce TNF-alpha and biweekly exposures by intravenous (IV)injection of B6 splenocytes (9×10⁶ splenocytes IV). B6 splenocytes are alymphoid cell population with intact MHC class I and self peptidepresentation that survives only transiently in vivo due to rejection bythe host.

Additionally, four diabetic control NOD mice were maintained over thesame time period with only insulin treatment. All control NOD mice weremonitored every other day for hyperglycemia and insulin was administereddaily unless normoglycemia returned. After approximately 40 days oftreatment, all control NOD mice receiving only insulin were dead. Poorblood sugar level control, cachexia, and weight loss accounted for theuniform mortality of all diabetic NOD hosts by day 20 (FIG. 4A). Controlmice treated only with insulin also had pancreatic histologydemonstrating impressive lymphoid infiltrates obscuring any recognizableislet structure (FIG. 4C).

In marked contrast, nine severely diabetic NOD mice receiving repeatexposures to B6 splenocytes plus TNF-alpha induction were alive in eightof nine cases and three of the NOD mice had returned to normoglycemia byday 40. In addition, four diabetic NOD mice treated with repeat B6splenocyte immunization and TNF-alpha induction, had improved islethistology by day 40 (FIG. 4D). Pancreatic islets were visible andlymphoid infiltrates were significantly reduced circumferentially aswell as adjacently to the islet structures. This pattern ischaracteristic of a histology pattern of protective, not destructive,lymphocyte infiltrates (Gazda et al., Journal of Autoimmunity (1997)10(3):261-70; Dilts et al., Journal of Autoimmunity (1999)12(4):229-32). Three diabetic NOD mice with TNF-alpha induction and B6splenocyte immunizations produced complete islet regeneration andinsulin independence. Histology on these three mice revealeddramatically reduced lymphocytic autoreactivity and increased isletabundance (FIG. 4D). Therefore, combined treatment with TNF-alphainduction and repeated exposure to peptide-bound B6 MHC class Ilymphocytes was sufficient to transiently obliterate autoreactive Tcells and reverse NOD diabetes for at least 40 days in approximately 30%of the hosts tested (FIG. 4). The therapy was partially protective inapproximately 50% of the NOD hosts (FIG. 4).

In order to eliminate poorly controlled blood sugar levels as a factorhampering more complete islet regeneration, additional groups ofdiabetic NOD mice were similarly treated, with TNF-alpha induction andB6 splenocyte injection, but with simultaneous implantation of aeuglycemic clamp intraperitoneally, for 40 days. A murine euglycemicclamp in these studies consisted of alginate encapsulated B6 islets. Thealginate capsule provides a membrane barrier system that allows shortterm glycemic control of insulin exchange but prevents direct cell-cellcontact (e.g., for T cell education). After 40 days, the euglycemic NODmice with the encapsulated islets underwent surgical removal of thealginate capsules and the blood sugar levels of the diabetic NOD micewere monitored for evidence of in situ pancreas regeneration.

Table 3, shows that after 40 days, both mice treated with the euglycemicclamp in the absence of TNF-alpha induction (Table 3, group 1) and micetreated with the euglycemic clamp and TNF-alpha induction (Table 3,group 2), showed an absence of endogenous islet regeneration and rapidlyreturned to hyperglycemia after clamp removal. Results indicate thatunder conditions of excellent glucose control and TNF-alpha induction,apoptosis of existing autoreactive cells is induced during the earlyphases of acute diabetes, but neither the degenerative state of thepancreas (as assayed by histology) nor the course of preexistingautoimmunity can be altered. The histology of NOD control treatmentgroups consisted of severe lymphocytic elimination of the islets in thepancreas (Table 3, FIG. 4).

TABLE 3 Impact of short-term (40 day) blood sugar control on endogenousislet regeneration in diabetic NOD mice after removal of euglycemicclamp # of normoglycemic Spleen cell TNF-alpha recipients Group donorinduction* Total # of recipients % 1 — − 0/7 0 2   + 0/6 0 3 B6 + 7/9 784 B6 class I^(−/−),^(−/−) + 2/6 33 5 C57 class II^(−/−), ^(+/+) +  8/1173 *Euglycemia was maintained for 40 days with an encapsulated isletallograft that was surgically removed on day 40. All the encapsulatedgrafts were removed on day 40 after transplantation. ¶Another fiverecipients rejected the encapsulated grafts before removal of the graftsprecluding the determination of euglycemia in islet regeneration.††C57-class II^(−/−) cells were from disruption of the Abb gene andexpresses no A or E MHC class II molecules and were purchased fromTaconic Research Laboratories (Germantown, NY). *TNF-alpha induction wasaccomplished with a single foot pad injection of CFA at the time of thefirst spleen cell injection of 9 × 10⁶ cells IV. †B6 classI^(− /−),^(−/−) donor splenocytes were from mice with both β₂M and Taplgene interruption.

In marked contrast, diabetic NOD mice that had received biweekly B6splenocyte immunizations in combination with a single dose of TNF-alphainduction therapy remained normoglycemic for 40 days after clamp removalin 78% of the cases. A total of nine diabetic NOD mice were treated withthis therapy and seven of the nine NOD mice had pancreatic histologythat demonstrated sustained and continuing islet regeneration days toweeks after euglycemic clamp removal (Table 3, FIG. 5). In general, hostislets had circumferential lymphocytic accumulations and in some caseswere aldehyde fuschin positive, (i.e., had excess insulin, beyond theamount to maintain normoglycemia).

Therefore, islet regeneration was optimized in established diabetic NODmice by maintenance of blood glucose levels (using a euglycemic clamp),ablation of autoreactive lymphocytes (by brief TNF-alpha induction), andrepeated exposure to MHC class I and peptide presenting cells, after a40 day course of bi-weekly B6 splenocyte injections. Furthermore, afterthe therapy was halted and autoimmunity eliminated, the immediate rescueand continuous expansion of the endogenous pancreas was sufficient forsustained tolerance to self antigens. The mechanism of splenocytere-education was defined as dependent upon the education complex of MHCclass I and endogenous peptides. As demonstrated in Table 3, injectionof splenocytes permanently ablated for MHC class I proteins (MHC classI^(−/−,−/−)) into diabetic NOD mice with euglycemic clamps led to poorin situ islet regeneration (Table 3, group 4, FIG. 5). Injection ofsplenocytes lacking MHC class II proteins (MHC class II^(−/−)) permittedin situ islet regeneration presumably due to continued expression ofendogenous peptide in the context of MHC class I (Table 3, group 5, FIG.5). Reestablishment of self tolerance and elimination of autoreactivitywas MHC class I dependent and MHC class II independent. The sustainedeffectiveness of this treatment is demonstrated in FIG. 5. Blood sugarmaintenance was observed beyond 20 days after the removal of theeuglycemic clamp.

Example 5 Regeneration of the Endogenous Pancreas

Generally, the percentage of CD3⁺ T cells in young NOD mice (<12 weeksof age) is low, but after 30 weeks of age the percentage of CD3⁺ T cellsincreases dramatically and exceeds that of control mice. In order toevaluate the impact of successful pancreas regeneration and rescue onNOD lymphocyte selection, flow cytometric analysis was performed on CD3⁺T cells from treated NOD mice.

Splenic CD3⁺ T cell percentages were evaluated in 5 treated NOD micereceiving various treatments; represented in FIG. 6 at 5 to 26 daysafter treatment had stopped. An untreated age-matched NOD female mousetreated with insulin had 56% of splenocytes staining with anti-CD3antibodies. The age-matched B6 female mouse had 27% positive splenocytes(FIG. 6), a trend previously reported (Miyazaki Clin. Exp. Immunol.85:60,622; Pontesilli Clin. Exp. Immunol. 97:70,84). Two mice weresuccessfully treated through either B6 or B6 class II^(−/−) splenocyteimmunizations, in conjunction with TNF-alpha induction, and displayedpancreas rescue and regeneration. Remarkably, both successfully treatedNOD mice had 40% of splenocytes staining with anti-CD3⁺ antibodies (FIG.6). In marked contrast, unsuccessfully treated age matched NOD mice(treated with only TNF-alpha induction therapy or TNF-alpha inductiontherapy in conjunction with B6-MHC class I^(−/−) splenocytes) had noalterations in the high number of splenic CD3⁺ cells (FIG. 6).Therefore, the impact of halted autoimmunity and re-establishment oftolerance was systemic and included markedly altered T cell selectionthat partially normalized numbers of CD3⁺ T lymphocytes in the spleen.These observations were further affirmed by my results which showed that(1) successful therapy results in a substantial reduction in naive Tcells expressing CD45RB^(high), CD62L, and CD95 proteins and (2) anincrease in the number of long-term memory cells, both indications ofthe normalization of T cell selection as a result of the combinationtherapy.

Example 6 Identification and Elimination of Autoimmune Cells

TNF-alpha sensitivity, in the NOD mouse, results from signaling defectsin the NF_(κ)B/proteasome activation pathway, thereby permitting theTNF-alpha combination treatment disclosed herein to be successful.Accordingly, the methods disclosed herein are applicable to any numberof mammalian systems. For example, I have shown that TNF-alpha inducedapoptosis also occurs in humans. In my studies using fresh humanlymphocytes, from patients with diverse autoimmune diseases, use of thecombination of CFA and MHC class I donor cells resulted in TNF-alphainduced apoptosis of autoreactive lymphocytes. Lymphocytes from patientswith type I diabetes (n=80), multiple sclerosis (n=5), and rheumatoidarthritis (n=10), were treated in vitro with 10-20 ng/ml of TNF-alphafor 24 hours and then evaluated for cell viability. Control lymphocytesfrom normal human donors were also studied (n=80). In all cases, thecontrol cells were found to be protected from cell death. In contrast,all lymphocytes from autoimmune samples showed varying degrees of celldeath after TNF-alpha exposure. These results support theabove-described notion that humans with diverse autoimmune diseases,similar to the NOD mice, have intracellular defects in signalingpathways such as NF_(κ)B, preventing normal viability after exposure tothis cytokine, TNF-alpha. Additional data from this study of humans hasrevealed that the severity of the autoimmune cell death after TNF-alphatreatment relates to the rate of onset of the disease not to theduration of the disease. This means, for example, that early onset ofdiabetic autoimmunity in a four-year old child is associated with agreater fraction of lymphocytes dying after TNF-alpha treatment than isobserved, following the same treatment, in lymphocytes from atwenty-year old person with onset of autoimmunity in early adulthood.Accordingly, it is likely that more aggressive autoimmune diseases,e.g., ones that appear earlier during a patient's lifetime, show agreater fraction of autoimmune cells susceptible to the treatment forautoimmune disease reversal as is discussed herein.

Example 7 Treatment of Sjorgen's Disease, RA, and Lupus

As detailed above, the therapeutic methods disclosed herein are readilyapplicable to any number of autoimmune diseases. For example, when aTNF-alpha treatment regimen combined with exposure to MHC class I-selfpeptide comlexes was applied to other forms of autoimmune disease, suchas murine models of Sjogren's disease, lupus symptomatology, andrheumatoid arthritis, I determined that autoimmune infiltratesindicative of Sjogren's disease were eliminated in the salivary andlachrymal glands, auto antibodies indicative of lupus were eliminated,and in a model of rheumatoid arthritis (RA), treatment of female RA micewith the therapy of this application halted limb swelling. The endpointof a therapy effectiveness was monitored by gross inspection, histologyof the target organ, and serology. With respect to RA, after diseaseonset, nearly 90% of the mice with severe RA were successfully treatedand the accompanying joint disease was no longer detectable by grossinspection or by histologic evidence at the time of death.

Example 8 In Vitro Monitoring

Treatment Scenario II

Prescreening: A human subject presenting symptoms of type I diabeteswill be brought into the clinic to give a single blood donation thatwill be divided into two tubes. One tube will be used to screen for thepresence of autoantibodies and the other tube will be used in an invitro screen for apoptotic cell death (e.g., TNF-induced) or acceleratedcell death due to any environmental or chemical agent. This initialsample will be used to obtain a base line C-peptide level and to verifythe absence of functional islets. Heightened in vitro sensitivity tocell death by any apoptotic cell death pathway will be a prerequisite tofuture therapy.

Treatment of Juvenile Onset Diabetes: If autoantibodies to islet cellsare present, we will conclude that the pancreas is still attempting toregenerate. Thus, if existing autoimmunity was eliminated by treatment,the islets could successfully regrow.

An inexpensive approach to try to immediately rescue the pancreas wouldbe to repeatedly perform BCG administrations, as a non-specificimmunostimulant that could successfully raise the levels of endogenousTNF-alpha activity. Endogenous TNF-alpha will kill only the autoimmunecells (i.e., cells with a defect in protection from apoptosis).Initially we will start out with weekly BCG immunization. Blood sampleswill be collected within 24-48 hours after BCG immunization and testedin vitro (in cell culture) for the persistence of TNF-alpha sensitiveautoimmune cells. The isolated cells, grown in cell culture, will beexamined to determine whether the autoimmune cells, sensitive to death,are eliminated or reduced by this administration.

If the response to BCG immunization is positive, we will then startimmunization with donor lymphocytes. Ideally, these lymphocyteimmunizations could be from both parents and would involve weeklyinfusions into the diabetic child, to be administered simultaneouslywith the BCG immunizations. In some cases, the donor lymphocytes will beirradiated to decrease the risk of infection transfer. We will introducethe lymphocytes intravenously at a dose of approximately 9×10⁶ to 9×10⁹as a start. With the in vitro monitoring assay, we expect to be able toidentify an optimal dose. Our goal is to continue this treatment from aminimum of 40 days up to about 6 months, or until positive human Cpeptide is found in the blood and insulin dosing is reduced. Early signsof possible tolerance induction success might not only be the presenceof C peptide but also the absence of TNF-alpha sensitive (autoreactive)cells. This would be an indication that the re-education is complete.Also, we predict that the phenotype of peripheral lymphoid cells wouldpossibly convert to a more mature phenotype (shown by an decreasedCD45RA to CD45RO ratio). Thus, screening in the lab will involve celldeath assays and lymphocyte surface markers of improved T cellselection. We expect also to see gross changes, such as reselection ofperipheral T cells and gross numbers of CD3 cells decreasing due toreintroduction of lymphoid cells directly, or due to regeneration ofislets that re-select peripheral T cells.

Treatment of Adult Onset Diabetes: If the patient is 40 years old andhas had diabetes for 20 or more years, we will follow the same treatmentprotocol, but extend treatment over a longer period of time. In longstanding disease, it is possible that the islet precursor cells of thepancreas are effectively inactive and can no longer multiply because ofyears of autoimmune attack. This treatment might protect the patientfrom complications of the disease, which in some cases may be directlyrelated to the altered cytokines of the poorly selected lymphoid cellscausing fibrosis. In addition, this treatment might eliminate theautoreactive cells that cause fibrosis and other complications. Lastly,this treatment might allow, for the first time, for islets to betransplanted with the barrier being only islet survival, not isletsurvival from graft rejection or islet survival against autoimmunity.

Treatment Scenario II

Subjects. Patients who are older than age 18 but younger than 45 yearsand who have Type 1 diabetes (insulin dependent and ketosis prone) willbe recruited for this study. Participants will have to have a durationof Type 1 diabetes, dated from the time of insulin administration, of atleast one month, but not more than 5 years. The rationale for theduration criterion is predicated on the expectation that persons withless than 5 years duration will still have residual beta cell mass whichis capable of recovery. Patients will be screened to determine whetherthey have autoantibodies (anti-GAD and anti-IA-2 or islet cellantibodies) present. In addition, the presence of functioning beta-cellmass, as measured by detectable (>0.2 pmol/ml) C-peptide levels afterglucagon stimulation, will be determined, although it will not be arequirement for inclusion in the study. Exclusion criteria will includepersons who have had previous BCG vaccination, a history of clinicaltuberculosis, or positive PPD test, a positive response to anintermediate (5 IU) PPD test, or any acute or chronic skin conditions.

Study Procedures: Eligible volunteers, as judged by chart review, willbe asked to come to the Diabetes Research Center where they will havemeasured both fasting and stimulated (6 min after 1 mg glucagon givenIV) C-peptide (endogenous insulin) secretion. In addition, blood sampleswill be obtained to measure autoantibody status (see above) and thelevel of TNF and autoreactive T-lymphocytes or peripheral lymphoid cellswith apoptosis sensitivity. Finally, if no recent (within four weeks)hemoglobin A1c level is available, one will be obtained. A standardpanel of liver function tests and a CBC with differential will also beobtained.

TNF-alpha induction, for example, by CFA administration or BCG“vaccination,” will be performed with a standard method with apercutaneous injection of 0.3 ml of a 50 mg/ml solution(Organon)(equivalent to a TNF-alpha induction of 10 μg/m²) into thedeltoid area. After the BCG solution is topically applied to the skin, asterile multipuncture disc is used to administer to BCG percutaneously.

Volunteers will be asked to return at weekly intervals for four weeks tohave a blood specimen obtained to repeat measurements of TNF andauto-reactive lymphocytes (1 green top and 1 red top tube). Thevaccination site (deltoid area) will be examined on each of theseoccasions to determine whether any significant ulceration or localreaction has occurred. In addition, patients will be questioned withregard to any febrile or other systemic symptoms that may have occurred.After four weeks, the patients will have a repeat vaccination performedin the opposite arm and similar, weekly monitoring will go on foranother two months.

Depending on the results from the first group of 5 subjects, adjustmentsin dosage and/or frequency of BCG vaccination will be made for asubsequent group of 5 individuals.

Risks: The risks entailed in this study are minor and include the minordiscomfort of obtaining blood samples. The total volume of bloodobtained over the course of the three-month study will be considerablyless than usually given in a single blood donation. The glucagonstimulated C-peptide test is commonly used in experimental protocols.The glucagon injection may be associated with mild nausea which usuallydissipates in 5 minutes. Rarely (less than 1 in 20) subjects may vomitafter glucagon.

BCG vaccination have been used for more than 30 years in many countries,including Canada and in western Europe, as a vaccination againsttuberculosis. The recognized side effects of BCG vaccination includemild local discomfort at the vaccination site with a papular rashdeveloping at the site 10-14 days after vaccination and reaching amaximal diameter of 3 mm 4-6 weeks after vaccination. The rash may scalethereafter, and rarely leaves a visible scar. Local adenopathy is rarelyseen in children, but almost never in adults. Rare events includeosteomyelitis, lupoid reactions, disseminated BCG infections and death.The frequency of these severe reactions is between 1 in 1,000,000 and 1in 5,000,000 vaccinations, and have occurred almost exclusively inimmunosuppressed children. Most of the recent experience with BCG hasbeen in the intravesicular treatment of bladder cancer, where weeklyinstallations of BCG are performed for ≧6 weeks. Finally, BCGvaccination has been used in Type 1 diabetes without any adverseconsequences noted.

All references cited herein are hereby incorporated by reference intheir entirety.

1. A method of increasing or maintaining the number of functional cellsof a predetermined type in a mammal, said method comprising the stepsof: a) providing a sample of cells of said predetermined type, b)treating said cells to modify the presentation of an antigen of saidcells that is capable of causing an in vivo autoimmune cell-mediatedrejection response, c) introducing said treated cells into said mammal,and d) prior to, after, or concurrently with step c), treating saidmammal to kill or inactivate autoimmune cells of said mammal.
 2. Themethod of claim 1, wherein said mammal is a human patient.
 3. The methodof claim 2, wherein said cells are insulin-producing islet cells.
 4. Themethod of claim 1, wherein step b) comprises eliminating, reducing, ormasking said antigen.
 5. The method of claim 1, wherein step d)comprises administering to said mammal TNF-alpha or a TNF-alpha inducingsubstance.
 6. The method of claim 5, wherein the TNF-alpha inducingsubstance is tissue plasminogen activator, LPS, interleukin-1, UV light,or an intracellular mediator of the TNF-alpha signaling pathway.
 7. Themethod of claim 1, wherein said mammal has a mutation in the lmp2 geneor equivalent thereof.
 8. A method of increasing the number offunctional cells of a predetermined type in a mammal, said methodcomprising the steps of: a) treating said mammal with an agent thatkills or inactivates autoimmune cells of said mammal; b) periodicallymonitoring the cell death rate of said autoimmune cells; and c)periodically adjusting the dosage of said agent administered to saidmammal based on the monitoring of step b).
 9. The method of claim 8,wherein said agent comprises TNF-alpha, a THF-alpha inducing substance,tissue plasminogen activator, LPS, interleukin-1, UV light, or anintracellular mediator of the TNF-alpha signaling pathway.
 10. Themethod of claim 5, wherein step d) comprises administering to saidmammal two agents that increase TNF-alpha.
 11. The method of claim 8,wherein step a) comprises administering to said mammal two agents thatincrease TNF-alpha.
 12. A method for diagnosing an autoimmune disease orthe predisposition to said disease in a mammal, said method comprisingthe steps of: a) providing peripheral cells from a mammal, b) treatingsaid cells with a TNF-alpha treatment regimen, and c) detecting celldeath of said peripheral cells, wherein an increase in cell death, whencompared with control cells, is indicative of said mammal having anautoimmune disease or a predisposition to said disease.
 13. The methodof claim 12, wherein said peripheral cells comprise splenocytes, Tlymphocytes, B lymphocytes, or cells of bone marrow origin.
 14. Themethod of claim 12, wherein said mammal is a human patient.
 15. Themethod of claim 12, wherein said TNF-alpha treatment regimen comprisestreating said peripheral cell with TNF-alpha.
 16. A method of treatingan autoimmune disease in a mammal comprising administering to saidmammal a composition comprising Bacillus Calmette-Guérin (BCG) bypercutaneous injection, wherein the autoimmune disease is selected frommultiple sclerosis, scleroderma, Sjogren's disease, systemic lupuserythematosus, Grave's disease, hypothyroidism, Crohn's disease,colititis, an autoimmune skin disease, and rheumatoid arthritis.
 17. Themethod of claim 16, wherein said autoimmune disease is Sjogren'sdisease.
 18. The method of claim 16, wherein said autoimmune disease ismultiple sclerosis.
 19. The method of claim 16, wherein said autoimmunedisease is rheumatoid arthritis.
 20. The method of claim 16, whereinsaid autoimmune disease is systemic lupus erythematosus.
 21. The methodof claim 16, wherein said autoimmune disease is Crohn's disease.
 22. Themethod of claim 16, wherein said autoimmune disease is an autoimmuneskin disease.
 23. The method of claim 16, wherein said mammal is ahuman.
 24. The method of claim 16, wherein said method further comprisesadministering a substance selected from TNF-alpha, a TNF-alpha receptoragonist, complete Freund's adjuvant, LPS, fMet-Leu-Phe, CD28, CD40, Fas(CD95), p75TNF, lymphotoxin Beta-receptor, and an agonist of TNF-alphaconverting enzyme.
 25. The method of claim 24, wherein said substance isa TNF-alpha receptor agonist.
 26. The method of claim 25, wherein saidTNF-alpha receptor agonist is an antibody.
 27. The method of claim 16,wherein said treating comprises killing or inactivating autoimmunecells.
 28. The method of claim 27, wherein said autoimmune cells areautoreactive T-cells.
 29. The method of claim 16, wherein said mammal ispresenting symptoms of said autoimmune disease.
 30. The method of claim23, wherein said method comprises repeatedly administering saidcomposition to said human.